Cherry P. Fernandez

Identification and expression analysis of duck interleukin-17D in Riemerella anatipestifer infection.

Interleukin (IL)-17D is a proinflammatory cytokine with currently largely unknown biological functions. Here we provide the description of the sequence, bioactivity, and mRNA expression profile of duck IL-17D homologue. A full-length duck IL-17D (duIL-17D) cDNA with a 624-bp coding region was identified from the large intestine. duIL-17D shares approximately 94.7% identity with its chicken counterpart, which is also identified in this work. duIL-17D exhibits 62.6-68.4% and 52.1-53.1% identity with mammalian and piscine homologues. Recombinant duIL-17D promoted the expression of proinflammatory cytokines such as IL-6, IL-8, and IL-1β in duck embryo fibroblast cells. Very low levels of duIL-17D transcript were observed in healthy lymphoid tissues, including bursa, thymus, and spleen, while duIL-17D expression was relatively high in the heart. The duIL-17D expression profiles were examined in mitogen-stimulated splenic lymphocytes, as well as tissues affected by Riemerella anatipestifer infection. The levels of duIL-17D were mostly upregulated in mitogen-activated splenic lymphocytes but downregulated in the liver and spleen of R. anatipestifer-infected ducks. These results provide new insights into the roles of IL-17D in host protective immune responses to Riemerella infection, which can therefore lead to further studies of its biological functions in different disease models of ducks and other avian species.

Molecular cloning, characterization and mRNA expression of duck interleukin-17F

Interleukin-17F (IL-17F) is a proinflammatory cytokine that plays an important role in gut homeostasis. A full-length duck IL-17F (duIL-17F) cDNA with a 510-bp coding region was identified in ConA-activated splenic lymphocytes. duIL-17F is predicted to encode 166 amino acids, including a 26-amino acid signal peptide, a single N-linked glycosylation site, and six cysteine residues that are conserved in mammalian IL-17. duIL-17F shares 77.5% amino acid sequence identity with chicken IL-17F (chIL-17F), 37-46% with corresponding mammalian homologues, and 53.5% with the previously described duck IL-17A (duIL-17A). The duIL-17F transcripts were expressed in a wide range of untreated tissues; levels were highest in the liver and moderate in the thymus, bursa, kidney, and intestinal tissues. Expression levels of duIL-17F transcript were slightly up-regulated in ConA- and LPS-activated splenic lymphocytes but not in poly I:C stimulated cells. duIL-17F forms heterodimers with duIL-17A. Recombinant duIL-17F, like duIL-17A, induced IL-1β, IL-6, and IL-8 expression in duck embryonic fibroblasts (DEFs). duIL-17A, but not duIL-17F expression, was significantly up-regulated in the liver and spleen of Salmonella Typhimurium-infected ducks. Further analysis of the contributions of IL-17F to different Salmonella spp. or other disease models will be required to expand our understanding of its biological functions.

Identification of alternatively spliced isoforms of interleukin-2/15 receptor β chain in ducks.

Interleukin (IL)-2 and IL-15 receptor β (IL-2/15Rβ, CD122) play important roles in signal transduction for biological functions of IL-2 and IL-15. We found that ducks possess three different IL-2/15Rβ transcripts, a conventional form (duIL-2/15Rβ) and two variants. Comparisons between the cDNA and genomic sequences revealed that the two variants, duIL-2/15Rβ-d7 and duIL-2/15Rβ-d9, were novel spliced transcripts resulting from skipping exons 7 and 9, respectively. Expression profiles of duIL-2/15Rβ and its isoforms were examined in healthy tissues, concanavalin A (ConA)-stimulated splenic lymphocytes and in livers and spleens of Riemerella anatipestifer-infected ducks using quantitative real-time PCR (qRT-PCR). Generally, duIL-2/15Rβ-d9 expression was undetectable in healthy tissues, ConA-activated samples, and R. anatipestifer-infected ducks. Expression levels of duIL-2/15Rβ transcript were relatively high to moderate in all healthy tissues tested, while duIL-2/15Rβ-d7 expression was low. Compared to untreated controls, expression levels of duIL-2/15Rβ were elevated in ConA-activated splenic lymphocytes and in livers on day 7 in R. anatipestifer-infected ducks, while duIL-2/15Rβ-d7 expression was unchanged. Additionally, COS-7 cells transfected with duIL-2/15Rβ, duIL-2/15Rβ-d7, or duIL-2/15Rβ-d9 constructs generated 73 kilodalton (kDa), 31kDa, and 40kDa proteins, respectively. This study identified three different IL-2/15Rβ transcripts, including two isoforms generated by alternative splicing and their gene expression patterns in stimulated conditions.

Identification of duck IL-4 and its inhibitory effect on IL-17A expression in R. anatipestifer- stimulated splenic lymphocytes

&NA; As the dysregulation of IL‐17 is implicated in the pathogenesis of various autoimmune and inflammatory diseases, the suppression of IL‐17 production by Th2 cytokines could alleviate the development of these diseases. Previously, we confirmed that inflammatory cytokines including IL‐17A are strongly associated with R. anatipestifer infection, which is one of the most important bacterial pathogens in the duck industry. Here, we found that IL‐4 treatment downregulated the expression of IL‐17A and IL‐17F transcripts in splenic lymphocytes stimulated with R. anatipestifer. Moreover, duck IL‐4 (duIL‐4) treatment in R. anatipestifer‐stimulated lymphocytes suppressed the expression of IL‐23p19 and IL‐12p40 transcripts compared to untreated and stimulated lymphocytes. Conversely, duIL‐4 increased levels of IFN‐&ggr; and IL‐10. We identified a full‐length duIL‐4 cDNA encoding 136 amino acids from ConA‐activated splenic lymphocytes that shares 49.3–50% amino acid sequence identity with chicken and quail IL‐4 and 21–29.7% with mammalian and piscine homologues. Low or moderate levels of duIL‐4 transcript were observed in healthy tissues, including the spleen, bursa, and thymus, whereas duIL‐4 expression was higher in the kidney and lung. Levels of duIL‐4 were generally upregulated in mitogen‐activated splenic lymphocytes but lower in the liver and spleen of R. anatipestifer‐infected ducks compared to those of infected chickens. Recombinant duIL‐4 promoted nitric oxide synthesis in duck macrophages stimulated by R. anatipestifer compared to untreated and stimulated control macrophages. These results demonstrate that IL‐4 is an important Th2 cytokine that inhibits inflammatory responses in splenic lymphocytes stimulated with R. anatipestifer.

Different strategies for producing naturally soluble form of common cytokine receptor γ chain

The common cytokine receptor γ chain (γc) plays an essential role in regulating lymphoid homeostasis. In fact, alteration of this gene causes severe immunodeficiency in humans and animals. Although soluble γc (sγc) was identified in the late 1990s, much remains unknown about its production. This study describes various mechanisms underlying the generation of sγc isoforms in different species. Our data demonstrate that mouse γc and the avian ortholog γc-a did not generate sγc. Moreover, two mouse isoforms, CRA-a and mγc-b, encoded by transcripts lacking a transmembrane region by alternative splicing, did not yield sγc. However, in ducks, sγc was produced from a γc-b transcript lacking a transmembrane region by alternative splicing. In chickens, sγc was produced in normal cells and cell lines by proteolytic shedding of the γc-b isoform containing intron 5, which displayed a relatively high probability of proteolytic cleavage of the ectodomain. This shedding was suppressed by leupeptin, serine and cysteine protease inhibitor. Compared to the chicken ortholog γc-a, expression of γc-b mRNA was differentially regulated according to tissue type, developmental stage, and antigen stimulation. These data demonstrate several mechanisms for producing sγc and suggest a potential role for sγc in avian lymphoid homeostatic responses to environmental antigens.

Downregulation of common cytokine receptor γ chain inhibits inflammatory responses in macrophages stimulated with Riemerella anatipestifer

&NA; Th17‐cell‐mediated inflammation is affected by the soluble form of common cytokine receptor &ggr; chain (&ggr;c). We previously suggested that inflammatory cytokines including interleukin (IL)‐17A are associated with Riemerella anatipestifer infection, which a harmful bacterial pathogen in ducks. Here, the expression profiles of membrane‐associated &ggr;c (du&ggr;c‐a) and soluble &ggr;c (du&ggr;c‐b) in R. anatipestifer‐stimulated splenic lymphocytes and macrophages, and in the spleens and livers of R. anatipestifer‐infected ducks, were investigated. In vitro and in vivo results indicated that the expression levels of both forms of &ggr;c were increased, showing that marked increases were detected in the expression of the du&ggr;c‐b form rather than the du&ggr;c‐a form. Treatment with &ggr;c‐specific siRNA downregulated mRNA expression of Th17‐related cytokines, including IL‐17A and IL‐17F, in duck splenic macrophages stimulated with R. anatipestifer, whereas the expressions of interferon (IFN)‐&ggr; and IL‐2 were enhanced. The results showed that the upregulation of &ggr;c, especially the du&ggr;c‐b form, was associated with expression of Th17‐related cytokines during R. anatipestifer infection. HighlightsTh17‐mediated inflammation is affected by soluble common cytokine receptor &ggr; chain (&ggr;c).Expression of membrane and soluble &ggr;c increased in R. anatipestifer‐infected ducks and stimulated lymphoid cells, especially marked expression of the soluble form.&ggr;c‐specific siRNA downregulated mRNA expression of Th17‐related cytokines IL‐17A and IL‐17F in R. anatipestifer‐stimulated macrophages.Upregulation of &ggr;c, especially du&ggr;c‐b (soluble) form, was associated with the expression of Th17‐related cytokines during R. anatipestifer infection.